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Fig. 1. Expression of JAK1 mRNA in testis. Reverse transcription
using total testis RNA as a template was carried out with (RT+)
or without (RT–) transcriptase. cDNA fragments encoding JAK1,
Xtr, and actin were amplified by polymerase chain reaction
(PCR), followed by the electrophoresis on 1% agarose gel and
staining with ethidium bromide.
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Fig. 2. Images of spermatogenic cells in
the section. The sections fixed with Bouinâs
fixative (A and Aâ) and 4% paraformaldehyde
in phosphate-buffered saline
(PBS) (B and Bâ) were immunostained with
anti-Xtr antiserum (Aâ and Bâ), followed by
staining with hematoxylin and eosin (A
and B). a, aâ, b, and bâ are enlarged
images of the areas surrounded by the
rectangles in A, Aâ, B, and Bâ, respectively.
PG, primary spermatogonium; SG,
secondary spermatogonia; PC, primary
spermatocytes; RT, round spermatids;
Scale bars indicate 50 lm in A, Aâ, B, and
Bâ; and 20 lm in a, aâ, b, and bâ.
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Fig. 3. Expression of JAK1 mRNA in
adult testis. The sections of the testis fixed
with 4% paraformaldehyde in phosphatebuffered
saline (PBS) were treated with
antisense (A) or sense (C) probe for JAK1
mRNA. After detection of the signals, the
sections were stained with hematoxylin
and eosin (A’, C’). A section adjacent to
the antisense probe-treated section was
immunostained with anti-Xtr antiserum to
detect spermatogenic cell (B). a, a’, and b
are enlarged images of the areas surrounded
by the rectangles in A, A’ and B,
respectively. PG, primary spermatogonium;
SG, secondary spermatogonia; PC,
primary spermatocytes; RT, round spermatids;
MS, mature sperm. Note that PGs
are distinguished by the presence (arrowheads
in A and a) or absence (double
arrowhead in A and a) of JAK1 mRNA.
Scale bars indicate 50 lm in A, A’, B, C
and C’; and 20 lm in a, a’, and b.
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Fig. 4. Effect of AZD1480 on the efficiency of BrdU-incorporation
into spermatogenic cell. After cultivation of the testes fragments
successively in follicle-stimulating hormone (FSH)-
supplemented medium with (5, 10, and 20 lmol/L) or without
(0 lmol/L) AZD1480 for 2 days and in the same medium containing
BrdU for 6 h, the number of primary spermatogonia (PG)
and cysts consisting of secondary spermatogonia (SG) with or
without BrdU in their nuclei was counted and their proliferating
activity was assessed by calculating the percentage of BrdUpositive
PG and SG. Each data represents a mean of three independent
experiments (PG: 0 lmol/L n = 18, 16, 18; 5 lmol/L
n = 22, 15, 8; 10 lmol/L n = 15, 13, 14; 20 lmol/L n = 20, 19,
23 and SG: 0 lmol/L n = 6, 7, 6; 5 lmol/L n = 8, 6, 7; 10 lmol/
L n = 8, 7, 10; 20 lmol/L n = 7, 6, 6) with a standard deviation
indicated by a bar. The difference between two values was analyzed
by Student’s t-test and statistical significance was shown
as P-value <0.01 (**) or 0.05 (*).
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Fig. 5. Expression of JAK1 mRNA in
juvenile testis. A and A’: Section of the
testis was treated with antisense probe for
JAK1 mRNA (A). After detection of the
signals, the section was stained with
hematoxylin and eosin (A’). Note that primary
spermatogonia (PGs) are distinguished
by the presence (arrowheads) or
absence (double arrowheads) of JAK1
mRNA. Scale bars indicate 20 lm. B:
Each isolated primary spermatogonium
(PG) and secondary spermatogonium (SG)
from the juvenile testis was examined for
JAK1 mRNA by single cell Reverse transcription-
polymerase chain reaction and
each band was examined for whether
specific DNA fragments had been amplified
by Southern hybridization. Note that
two (lanes 2 and 6) out of nine PGs did
not express JAK1 mRNA. Xtr, germ cell
marker, and actin were also run as internal
controls.
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Fig. 6. Expression of JAK1 mRNA in spermatogonial stem cells.
By single cell reverse transcription–polymerase chain reaction
(RT–PCR), the presence of JAK1 mRNA was examined in each
isolated spermatogonial stem cell (SSC) from the juvenile testes
that were cultured in a medium supplemented with follicle-stimulating
hormone for 2 weeks. Note that five (lanes 2, 4, 6, 7, and
8) out of 10 SSCs did not express JAK1 mRNA. Xtr, germ cell
marker, and actin were also run as internal controls. Each bands
was examined for whether specific DNA fragments had been
amplified by Southern hybridization.
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Fig. 7. Expression of JAK1 mRNA in regenerating testis. (A) A
5.0 mm long adult testis before surgery. (B) The same testis shortened
to 2.2 mm by partial removal. (C) The same testis recovered
to 5.5 mm long at 5 weeks after the surgery. Scale bars indicate
2 mm. (D) The ratio of PGs without JAK1 mRNA in the testes
before and after induction of regeneration were determined by
in situ hybridization using antisense probe for JAK1 mRNA. Each
data represents a mean of three independent experiments (before:
n = 200, 213, 155 and after: n = 213, 240, 220) with a standard
deviation indicated by a bar. The difference between the two
values (“before†and “afterâ€) was analyzed by Student’s t-test. The
P-value obtained was <0.01 (**) and statistically significant.
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Fig. 8. Asymmetric distribution of JAK1 mRNA in barrel-shaped primary spermatogonia (PGs). Using antisense RNA probe for JAK1 mRNA,
JAK1 mRNA in the PGs was detected by in situ hybridization on the sections from Cytochalasin B-treated adult testis (A’, Aâ€, B’, and Bâ€)
and Cytochalasin B-treated regenerating adult testis (C’ and D’). The blue signal indicating the presence of JAK1 mRNA was detected only
in one side of the barrel-shaped PGs (arrowheads; A’, Aâ€, B’, and Bâ€) or not detected at all (C’ and D’). The double arrowheads indicate a
side with no signal. A’ and B’ are higher contrasted images of A†and Bâ€, respectively. After detection of JAK1 mRNA by in situ hybridization,
the sections were stained with propidium iodide (A, B, C, and D). D and D’ are enlarged images of the areas surrounded by the rectangles
in C and C’, respectively. The barrel-shaped PGs are outlined by the dotted lines (A, B, and D). Note that the barrel-shaped PGs possessed
two nuclei having weak stainability with propidium iodide. Scale bars indicate 10 lm in A, A’, Aâ€, B, B’, Bâ€, D, and D’; 20 lm in C and C’.
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Fig. 9. The hypothetical model of the timing of expression and
asymmetric distribution of JAK1 mRNA in spermatogenic stem
cells (SSCs). The SSC preparing for production of primary spermatogonium
(PG) destined to differentiate (Differentiation) but not
the self-renewing SSC (Self-renewal) expresses JAK1 mRNA (red
dots). The accumulated JAK1 mRNA is distributed exclusively
into the PGs destined to differentiate into sperm (Destined PG)
during the SSC division.
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